Int.J.Curr.Microbiol.App.

Sci (2017) 6(3): 1046-1051

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 1046-1051
Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2017.603.121

Explicit Effect of Phyllospheric Microorganism on Growth Promotion of

Pearl Millet (Pennisetum glaucum)
Deepika Chaudhary1*, Rakesh Kumar1, Anju Kumari2, Rashmi1 and Raman Jangra1
1
Department of Microbiology, CCS Haryana Agricultural University, Hisar, India
2
Center for Food Science and Technology, CCS HAU Hisar, India
*Corresponding author

ABSTRACT

Keywords
The present investigation aims to explore potential of phyllospheric isolates
Pearlmillet, Foliar, on pearlmillet. Bacteria from leaves were isolated and tested for their plant
Spray, Growth,
PGPR.
growth promoting traits i.e., siderophore production, indole acetic acid,
ammonia production and zinc solubilization. Three bacterial isolates were
Article Info selected and studied in pothouse on pearlmillet by foliar application. Isolate
Accepted: JFS-5 foliar spray exhibited maximum plant height and shoot dry weight.
18 February 2017 These promising results conclude that bacterial isolates from phyllosphere
Available Online:
12 March 2017
and their foliar applications can increase plant growth.

Introduction
Fertilizers are used in large amount by
farmers to increase crop production.
However, frequent and excessive use of these
chemicals has often resulted in adverse
environmental effects, disturbing the
ecological balance and making plants even
more susceptible to pests and diseases
(Olajire et al., 2015). Public awareness to
these problems has shifted the approach
towards some alternative measures. The use
of microorganisms is very important as
biofertilizers. They are capable to promote
plant growth, sustain soil productivity and soil
health. The foliar microbial population can
have positive, neutral, or negative impact on
their hosts (Berlec, 2012; Vorholt, 2012;
Bulgarelli et al., 2013; Rastogi et al., 2013).
The phyllosphere bacteria have significant
effects in the field of agriculture and
environment. These may affect the fitness of
natural plant populations and quality as well
as productivity of agricultural crops.
However, there has been little study of
bacterial communities on the surface of plants
(Kim et al., 2012). The objective of the
present investigation was to study the effect
of foliar applications of selected isolates on
plant growth parameters of pearlmillet in pot
house conditions.
Materials and Methods
Isolation from leaves of pearlmillet
Leaves sample were collected from CCS
HAU farm field during June-July (2014) from

1046
 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1046-1051
pearlmillet crop. Isolation of free living
diazotrophs was done by using serial dilution
method (Lindow et al., 1978) on Jenson’s
nitrogen free medium. Ten gram of leaf
sample was chopped and immersed in 100 ml
of sterilized water and shaken for 30 minutes.
Leaf washing thus obtained, were serially
diluted with sterile distilled water, these
plated on JM plates and incubated at 30oC for
6 days. Isolated colonies were purified,
maintained on JM slants and stored at 4oC.
Isolates were transferred to fresh JM slants at
regular intervals. Composition of Jensen’s
medium: (g/l, pH 7); sucrose 20.0,
K2HPO41.00, MgSO4. 7H2O.50, NaCl.50,
CaCl2.10, Na2MoO4.2H2O.005,
FeSO4.7H2O.050 and Agar-agar 20.0g/l in
1000 ml distilled water (Jensen 1951).
Plant growth promoting attributes
The functional diversity amongst isolated
Azotobacter strains was studied by qualitative
screening of their plant growth promoting
(PGP) attributes. The production of
Siderophore production by the isolates was
measured on Chrome Azurol-S (CAS) agar
(Schwyn and Neilands, 1987), by measuring
the diameter of the zone of the color change
from bluish green to orange. Quantitative
estimation of the indole acetic acid (IAA) was
carried out by inoculating 1 ml of the
respective bacterial suspension (07CFU ml−1)
in 10 ml Luria Bertani (LB) broth containing
L-tryptophan (100 μg ml−1), and incubating it
in the dark for 48 h. The concentration of IAA
in the culture supernatant was estimated by
the procedure of Gordon and Weber (1951).
All the isolates were tested for their ability to
excrete ammonia by growing the cultures in
30 ml JM broth. The flasks were incubated at
300C for 12 days under stationary conditions.
Aliquots of the culture broth were withdrawn
and centrifuged at 10,000 rpm for 15 min.
Ammonia released in the supernatant was
determined according to the method of
1047
Chaney and Marbach (1962). Solubilisation
of zinc were assessed according to the
procedure of Saravanan et al., (2007) using
nutrient agar medium containing 0.1 %
insoluble zinc compounds [ZnO, ZnS,
Zn3(PO4)2 and ZnCO3], respectively. Three
Azotobacter strains were selected for pot
house study on the basis of their growth
promoting attributes.
Foliar application of selected Azotobacter
strains on leaves of Pearlmillet
The effect of selected Azotobacter strains on
the growth of Pearlmillet (Pennisetum
glaucum) var. HHB-67 was studied under pot
house conditions. Experiment was done with
three treatments using two doses of fertilizer
75% and RDF with control (without culture),
respectively. Selected three isolates were
grown in Jensen’s nitrogen free broth. These
cultures were centrifuged and cell pellets was
washed twice.
These washed cells were further used for
foliar applications in Pearl millet done after
30 days of germination. Plant height and
shoot dry weight were determined at 60 days
of sowing. Total bacterial and Diazotrophs
count were also determined after 30 days
(before foliar application) and 60 days of
sowing.
Results and Discussion
Phyllosphere bacterial communities have
potential to influence plant biogeography and
ecosystem function through their influence on
the fitness and function of their hosts. In this
study, total 14 bacterial isolates were obtained
from phyllosphere of pearlmillet crop on
Jensons nitrogen free medium Petriplates.
These all strains were studied for different
plant growth promoting attributes i.e., IAA,
Ammonia, Siderophore production and Zinc
solubalization.
 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1046-1051

Plant growth promoting attributes that might facilitate plant nutrition.

All 14 Azotobacter strains produced ammonia
and IAA while only 8 strains produced
siderophore. Out of 14 isolates, 7 strains
exhibited solubilisation of Zinc respectively
(Table 1). Among PGP activities, IAA and
Ammonia producing strains were highest (100
%) when compared to Siderophore production
(57.14 %), zinc solubilisation (50 %). Strain
JFS-5 showed highest IAA production (13.50
μg ml-1) and Ammonia excretion (4.70 μg ml-
1
). Highest solubilisation of Zinc was shown
by strain JFS-10 (39 mm). Siderophore
production maximum by JFC-1 (18 mm)
exhibited (Table 1). Enya and co-worker
(2007) studied that phyllospheric
Sphingomonas spp. also produce IAA. Along
with phytohormone production, plant growth
promotion is known to be mediated by a
variety of mechanisms including
solubilisation of phosphorus, potassium and
zinc, production of ammonia, siderophores
and HCN (Dursun et al., 2010; Eman et al.,
2011; Mwajita et al., 2013). Ferrara et al.,
(2012) observed that the endophytic
diazotrophs from sugarcane roots are able to
produce plant growth-promoting substances
and to secrete higher amounts of amino acids
Effect on pearl millet growth after spray
bioinoculants on leaves
Azotobacter strains isolated from leaves of
pearl millet were used for studies on Pearl
millet crop under pot house conditions. There
was significant increase in Plant height and
shoot dry weight of Pearl millet. Maximum
increase in plant parameters was observed
with Azotobacter strain JFS-5 at fertilization
dose of N P K. Maximum plant height (51
cm) and shoot dry weight (910 mg) was
observed with foliar spray of JFS-5 with
recommended dose of fertilizers followed by
JFC-1 plant height (48.66 cm) shoot dry
weight (890 mg) and JFS-10 over the control.
This reflects the positive effect of foliar
application of isolates (Fig. 1 and Fig. 2).
Studies on survival count of total bacterial
and diazotrophs population increased at 60
days of leaves sampling as compared to initial
observation before spray of bacteria on leaves
(Fig. 3). Isolate JFS-5 with RDF showed
maximum bacterial counts (cfu ml−1) at 60
days as compared to other isolates and
control.

Table.1 Production of different beneficial properties by phyllospheric isolates

Serial Isolates IAA (µg Ammonia Zinc solubilization Siderophore

No. ml-1) (µg ml-1) Zone Diameter production
(mm) (mm)
1. JFC1 8.05 0.10 32 18
2. JFL2 9.64 0.03 - -
3. JFL3 8.47 0.10 - -
4. JFS4 9.22 0.49 - -
5. JFS5 13.50 4.70 21 13
6. JFS6 10.76 0.80 26 11
7. JFS7 11.21 1.37 27 13
8. JFS8 11.72 2.36 - -
9. JFS9 12.81 4.02 - 10
10. JFS10 9.98 4.01 39 11
11. JFS11 7.59 4.52 31 14
12. JFS12 11.30 4.51 - 14
13. JFS13 11.66 4.59 10 -
14. JFS14 10.46 4.69 - -

1048
 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1046-1051

Fig.1 Effect of phyllospheric selected isolates on plant height of

pearlmillet after 60 days plant growth

Fig.2 Effect of phyllospheric selected isolates on shoot dry weight of

pearlmillet after 60 days plant growth

Fig.3 Survival count of bacteria on leaves of pearlmillet at 30 and 60 days

1049
 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1046-1051
Diazotrophs count was maximum showed
also by JFS-5 + RDF i.e., 5.78 log value of
cfu g-1. There was significant increase in the
population of bacteria on leaves those applied
bioinoculants in spray as compared to control
treatment.
Esitken et al., (2010) studied the effect of
bacteria as root inoculation and foliar spray
on leaves of strawberry cv. Fern. They found
that both root inoculation of Bacillus M3 and
spraying of Pseudomonas BA-8 or Bacillus
OSU-142 have potential to increase the yield,
growth and P, Fe, Cu and Zn content of the
strawberry plant and to increase the soil P, Fe,
Zn, K, and Mg availability. Root inoculation
of M3 and floral and foliar spraying of OSU-
142 and BA-8 bacteria stimulated plant
growth resulting in significant yield increases.
Saa et al., (2015) studied the effect of one
biostimulant derived from sea weed extraction
(Bio-1) and another biostimulant derived from
microbial fermentation (Bio-2) on 2-years-old
almond plants over two growing seasons in
different treatment i.e., two soil potassium
treatments (12511 μgg –of Kvs. 5 μgg−) and
four foliar treatments (No spray,Foliar-K,Bio-
1,Bio-2). Rubidium was utilized as a
surrogate for short-term potassium uptake and
plant growth, nutrient concentration, and final
plant biomass were evaluated. There was a
substantial positive effect of both biostimulant
treatments on total shoot leaf area, and
significant increases in shoot length and
biomass under adequate soil potassium. In
conclusion all foliar application of selected
Azotobacter isolates treatments has positive
effect on growth of pearlmillet crop. These
can be a promising alternative as foliar
biofertilizer for sustainable and organic
agriculture system to mitigate problems
associated with use of chemicals fertilizers.
References
Berlec, A. 2012. Novel techniques and
findings in the study of plant
1050
microbiota: search for plant probiotics.
Plant Sci., 193: 96–102.
Bulgarelli, D., Schlaeppi, K., Spaepen, S.,
Themaat, E. and Lefert, P. 2013
Structure and functions of the bacterial
microbiota of plants. Annual Review of
Plant Biol., 64: 807–838.
Chaney, A.L. and Marbach, E.P. 1962
Methods for evaluating biological
nitrogen fixation. In F. J. Bergersen
(ed.) Clinical chemistry 8: 130. John
Wiley and Sons, New York, USA.
Dursun, A., Ekinch, M. and Donmez, M.F.
2010. Effects of foliar application of
plant growth promoting bacterium on
chemical contents, yield and growth of
tomato (Lycopersicon esculentum L.)
and cucumber (Cucumis sativus L.).
Pak. J. Bot., 42: 3349-3356.
Eman, A., Wafaa, E.K. and Elsisi, A.A.Z.
2011. Effect of foliar application of
some growth promoters on growth,
fruiting and fruit quality of sultani fig
trees. J. Agri. Environ. Sci., 10: 1-10.
Enya, J., Shinohara, H., Yoshida, S., Negishi,
T.T.H., Suyama, K. and Tsushima, S.
2007. Culturable leaf-associated
bacteria on tomato plants and their
potential as biological control agents.
Microbial Ecol., 53: 524-536.
Esitken, A., Yildiz, H.E., Ercisli, S., Donmez,
M., Turan, M. and Gunes, A. 2010.
Effects of plant growth promoting
bacteria (PGPB) on yield, growth and
nutrientcontents of organically grown
strawberry. Scientia Horticulturae, 124:
62–66.
Ferrara, F.I.S., Oliveira, Z.M., Gonzales,
H.H.S., Floh, E.I.S. and Barbosa, H.R.
2012. Endophytic and rhizospheric
enterobacteria isolated from sugar cane
have different potentials for producing
plant growth-promoting substances.
Plant and Soil, 353: 409- 417.
 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1046-1051

Gordon, S.A. and Weber, R.P. 1951
Colorimetric estimation of indoleacetic
acid. Plant Physiol., 26: 192-195.
Jensen, V. 1951 Notes on biology of
Azotobacter. Proceeding Society for
Appl. Bacteriol., 74: 89-93.
Kim, M., Singh, D., LieHoe, A., Go, R.,
Rahim, A.R., Ainnuddin, A.N., Chun, J.
and Adams, M.J. 2012. Distinctive
phyllosphere bacterial communities in
tropical trees. Microbial Ecol., 63: 674-
681.
Lindow, S.E., Arny, D.C. and Upper, C.D.
1978. Distribution of ice nucleation-
active bacteria on plants in nature. Appl.
Environ. Microbiol., 36: 831-838.
Mwajita, M.R., Murage, H., Tani, A. and
Kahangi, E.M. 2013, Evaluation of
rhizosphere, rhizoplane and
phyllosphere bacteria and fungi isolated
from rice in Kenya for plant growth
promoters. Springer Plus, 2: 606.
Olajire, A.B.L., Dada, O.V., Waha, O.M. and
Ojo, O.L. 2015. Effects of fertilizers on
soils microbial growth and populations:
a review. American J. Engi. Res., 4: 52-
61.
Rastogi, G., Coaker, G.L. and Leveau, J.H.
2013. New insights into the structure
and function of phyllosphere microbiota
through high-throughput molecular
approaches. FEMS Microbiol. Lett.,
348: 1–10.
Saa, S., DelRio, A., Castro, S. and Brown, P.
2015. Foliar application of microbial
and plant based biostimulants increases
growth and potassium uptake in
almond. Frontiers in Plant Sci., 6: 1-9.
Saravanan, V.S., Madhaiyan, M. and
Thangaraju, M. 2007. Solubalization of
zinc compounds by the diazotrophic,
plant growth promoting bacterium
Gluconacetobacter diazotrophics.
Chemosphere, 66: 1794-1802.
Schwyn, B. and Neilands, J.B. 1987.
Universal chemical assay for the
detection and determination of
siderophores. Anal. Biochem., 160: 47-
56.
Vorholt, J.A. 2012. Microbial life in the
phyllosphere. Nature Rev. Microbiol.,
10: 828–840.

How to cite this article:

Deepika Chaudhary, Rakesh Kumar, Anju Kumari, Rashmi and Raman Jangra. 2017. Explicit
Effect of Phyllospheric Microorganism on Growth Promotion of Pearl Millet (Pennisetum
glaucum). Int.J.Curr.Microbiol.App.Sci. 6(3): 1046-1051.
doi: https://doi.org/10.20546/ijcmas.2017.603.121

1051